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Dengue Virus Infection Causes the Activation of Distinct NF-κB Pathways for Inducible Nitric Oxide Synthase and TNF-α Expression in RAW264.7 Cells.

Cheng YL, Lin YS, Chen CL, Wan SW, Ou YD, Yu CY, Tsai TT, Tseng PC, Lin CF - Mediators Inflamm. (2015)

Bottom Line: In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation.Heat-inactivated DENV failed to cause the identified inflammatory responses.Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.

ABSTRACT
Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression.

No MeSH data available.


Related in: MedlinePlus

ROS is independent of NF-κB activation followed by TNF-α and iNOS expression during DENV infection. (a) CM-H2DCFDA-based staining followed by flow cytometric analysis determined ROS generation in RAW264.7 cells infected with DENV 2 at the indicated time points. The mean fluorescence intensity (MFI) of each stain is shown as the mean ± SD of three individual experiments. ns: not significant. In the presence of the ROS-scavenger NAC, NF-κB reporter assay (b), ELISA (c), Griess reagent (d), and western blot analysis (e) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected cells. DMSO was used for the negative control. ∗∗P < 0.01 and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. ns: not significant. For western blot results, one set of representative data obtained from three independent experiments is shown. The relative ratio to β-actin based on densitometer quantification and analysis using ImageJ software is shown. For all experiments, the quantitative data shown represent the mean ± SD values of three independent experiments.
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fig4: ROS is independent of NF-κB activation followed by TNF-α and iNOS expression during DENV infection. (a) CM-H2DCFDA-based staining followed by flow cytometric analysis determined ROS generation in RAW264.7 cells infected with DENV 2 at the indicated time points. The mean fluorescence intensity (MFI) of each stain is shown as the mean ± SD of three individual experiments. ns: not significant. In the presence of the ROS-scavenger NAC, NF-κB reporter assay (b), ELISA (c), Griess reagent (d), and western blot analysis (e) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected cells. DMSO was used for the negative control. ∗∗P < 0.01 and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. ns: not significant. For western blot results, one set of representative data obtained from three independent experiments is shown. The relative ratio to β-actin based on densitometer quantification and analysis using ImageJ software is shown. For all experiments, the quantitative data shown represent the mean ± SD values of three independent experiments.

Mentions: One of the possible mechanisms modulating NF-κB activation is the ROS-mediated pathway [14]. We examined the possibility of ROS involvement in DENV-induced NF-κB activation followed by TNF-α, NO, and iNOS induction. DCFDA staining followed by flow cytometry was used to detect changes in the ROS level in a time-dependent manner. However, there was no significant difference after DENV infection at the indicated time points (Figure 4(a)). Inhibiting ROS using NAC did not affect DENV-induced NF-κB activation (Figure 4(b)). NAC also had no influence on TNF-α and iNOS expression (Figures 4(c) and 4(e)). However, inhibition of ROS reduced NO generation significantly during DENV infection (Figure 4(d)). These results indicate that ROS is not responsible for the induction of NF-κB activation followed by TNF-α and iNOS upregulation but plays some role in the conversion of iNOS to NO during DENV infection.


Dengue Virus Infection Causes the Activation of Distinct NF-κB Pathways for Inducible Nitric Oxide Synthase and TNF-α Expression in RAW264.7 Cells.

Cheng YL, Lin YS, Chen CL, Wan SW, Ou YD, Yu CY, Tsai TT, Tseng PC, Lin CF - Mediators Inflamm. (2015)

ROS is independent of NF-κB activation followed by TNF-α and iNOS expression during DENV infection. (a) CM-H2DCFDA-based staining followed by flow cytometric analysis determined ROS generation in RAW264.7 cells infected with DENV 2 at the indicated time points. The mean fluorescence intensity (MFI) of each stain is shown as the mean ± SD of three individual experiments. ns: not significant. In the presence of the ROS-scavenger NAC, NF-κB reporter assay (b), ELISA (c), Griess reagent (d), and western blot analysis (e) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected cells. DMSO was used for the negative control. ∗∗P < 0.01 and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. ns: not significant. For western blot results, one set of representative data obtained from three independent experiments is shown. The relative ratio to β-actin based on densitometer quantification and analysis using ImageJ software is shown. For all experiments, the quantitative data shown represent the mean ± SD values of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496656&req=5

fig4: ROS is independent of NF-κB activation followed by TNF-α and iNOS expression during DENV infection. (a) CM-H2DCFDA-based staining followed by flow cytometric analysis determined ROS generation in RAW264.7 cells infected with DENV 2 at the indicated time points. The mean fluorescence intensity (MFI) of each stain is shown as the mean ± SD of three individual experiments. ns: not significant. In the presence of the ROS-scavenger NAC, NF-κB reporter assay (b), ELISA (c), Griess reagent (d), and western blot analysis (e) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected cells. DMSO was used for the negative control. ∗∗P < 0.01 and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. ns: not significant. For western blot results, one set of representative data obtained from three independent experiments is shown. The relative ratio to β-actin based on densitometer quantification and analysis using ImageJ software is shown. For all experiments, the quantitative data shown represent the mean ± SD values of three independent experiments.
Mentions: One of the possible mechanisms modulating NF-κB activation is the ROS-mediated pathway [14]. We examined the possibility of ROS involvement in DENV-induced NF-κB activation followed by TNF-α, NO, and iNOS induction. DCFDA staining followed by flow cytometry was used to detect changes in the ROS level in a time-dependent manner. However, there was no significant difference after DENV infection at the indicated time points (Figure 4(a)). Inhibiting ROS using NAC did not affect DENV-induced NF-κB activation (Figure 4(b)). NAC also had no influence on TNF-α and iNOS expression (Figures 4(c) and 4(e)). However, inhibition of ROS reduced NO generation significantly during DENV infection (Figure 4(d)). These results indicate that ROS is not responsible for the induction of NF-κB activation followed by TNF-α and iNOS upregulation but plays some role in the conversion of iNOS to NO during DENV infection.

Bottom Line: In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation.Heat-inactivated DENV failed to cause the identified inflammatory responses.Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.

ABSTRACT
Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression.

No MeSH data available.


Related in: MedlinePlus